The mechanism of initiation of RNA synthesis by eukaryotis RNA polymerase II will be studied using Ad2 DNA. Polymerases isolated from wheat germ, calf thymus and human placenta will be used. We will try to determine whether these polymerases are able to bind to double stranded DNA and form stable binary complexes. If such complexes are formed experiments will be performed to test the existence of two types of complexes: closed complexes which are not able to initiate, and open complexes which can rapidly initiate. The possibility of a two step model of initiation for eukaryotic polymerase similar to the model proposed by Chamberlin for prokaryotic polymerases will be tested. The conditions of formation of such complexes, their stability and position on Ad2 DNA will be determined. The mapping of the positions will be done using the EM method of protein-free BAC spreading for visualization of the position of polymerase on DNA. The structure of a polymerase capable of forming binary complexes will be determined for all three polymerases in hopes of identifying the subunits responsible for this activity. The antibody against individual subunits will be prepared in order to further study the function of the subunits in the process of promoter selection. Finally we will analyze RNA obtained in vitro and compare it to that synthesized in vivo. This will determine fidelity of in vitro transcription. The understanding of the mechanism of initiation (promoter selection) by eukaryotic polymerase would significantly contribute to the understanding of regulation of gene expression in eukaryotic cells.